Recent evidence implicates cilia abnormalities as important in the etiology of Polycystic Kidney Disease. Our laboratory has defined an important role for polarity proteins, such as the apical transmembrane protein Crumbs3, in ciliogenesis. In the course of our study on Crumbs3, we have identified a novel cilia and centrosomal trafficking mechanism that involves the Ran/Importin system. Ran is a small GTPase that is concentrated in the nucleus and regulates the import of proteins into the nucleus in combination with Importins. Importins travel into the nucleus carrying their cargo and when they bind to RanGTP, the cargo is released leading to overall nuclear import. However Ran is also concentrated at other points in the cell including the centrosome. We have found that Importin beta1 can bind to a splice form of Crumbs3, called Crumbs3-CLPI, and target this isoform of Crumbs to the centrosome. We also have obtained evidence that another cilia protein, called RP2, also uses the Importin system for trafficking to the cilia. Our hypothesis that guides this proposal is that the Ran/Importin system plays a major role in regulation of cilia and centrosomal trafficking. To test this hypothesis, we propose three specific aims. The first specific aim will be to identify the molecular basis for the Crumb3-CLPI interaction with Importins and determine how mutations that alter this interaction affect the trafficking of this isoform of Crumbs3. The second specific aim will test the role of other Importins, including Importin alphas, in this trafficking mechanism and in cilia function in general. In the last specific aim we will manipulate the levels of RanGTP at the centrosome and determine the effect of this manipulation on centrosome and cilia trafficking. We will also work to identify Ran and Importin alpha binding proteins at the centrosome. At the conclusion of these studies we will have a greater understanding of the role of the Ran/Importin system in cilia trafficking networks. This will have important implications for our understanding of Polycystic Kidney Disease and other ciliopathies. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to study mechanisms that control the movements of proteins into a cell organelle called the cilia. Recent studies have suggested that defective function of the cilia leads to cystic diseases of the kidney. Cystic diseases of the kidney, where the normal kidney is replaced by non-functioning fluid filled cysts, are a major cause of kidney failure.